p70S6K promotes IL-6-induced epithelial-mesenchymal transition and metastasis of head and neck squamous cell carcinoma

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer worldwide and a common cause of cancer-related death, with a 5-year survival rate of less than 60%. IL-6 has been suggested to play an important role in cancer metastasis, but its mechanism in HNSCC has not been fully clarified. p70S6K has been reported to induce epithelial-mesenchymal transition (EMT) of ovarian cancer, but its role in HNSCC remains unknown. In this study, we found that p70S6K and IL-6 were upregulated in high-metastatic HNSCC cell lines that underwent EMT when compared to paired low-metastatic cell lines. Overexpression of p70S6K promoted EMT and migration of HNSCC cells, while downregulation of p70S6K attenuated IL-6-induced EMT and cell migration. Furthermore, IL-6-induced p70S6K activation was attenuated by inhibitors of the PI3K/Akt/mTOR, MAPK/ERK, and JAK/STAT3 signaling pathways, suggesting that it located downstream of these pathways. These findings suggest that p70S6K promotes IL-6-induced EMT and metastasis of HNSCC. Targeting p70S6K for HNSCC therapy may benefit patients through the inhibition of tumor growth, as well as metastasis.

Keywords: HNSCC; IL-6; epithelial-mesenchymal transition; metastasis; p70S6K.

Figure 1. IL-6 is upregulated in 686LN-M4e cells compared to 686LN cells, and IL-6 induces EMT and migration

(A) total mRNAs of 686LN and 686LN-M4e cells were prepared and subjected to qRT-PCR assay. Columns, mean of three replicate determinations; bars, SD. *p < 0.05. (B) 686LN and 212LN cells were serum starved overnight, then stimulated with different concentrations of IL-6, as indicated, for 48 hours. The whole-cell protein lysates were prepared and subjected to western blot analysis. (C) 686LN or 212LN cells were seeded into the chambers in the 24-well plates with serum-free medium. Then, the medium in the outside of the chamber was replaced with condition medium containing 50 ng/ml IL-6 or its vehicle for another 24 hours and subjected to transwell assay. Cells on the bottom side of the chamber were recorded under a microscope. Magnification: 100×.

Figure 2. p70S6K is upregulated in 686LN-M4e cells compared to 686LN cells, and IL-6 activates p70S6K

(A) 686LN cells and 686LN-M4e cells were seeded to 10 mm dishes for 24 hours. (B) 686LN cells were serum starved overnight, then treated with IL-6 50 ng/ml for different times, as indicated. Whole-cell protein lysates were prepared and subjected to western blotting.

Figure 3. p70S6K induces EMT and migration

(A) 686LN and 212LN cells were transfected with vector (pRK7) or p70S6K wild type constructs (p70S6K), as indicated, for 48 hours. Then whole-cell protein lysates were prepared and subjected to western blotting. The fold change of each treatment vs. the control was calculated after quantification and presented under each blot. (B) 686LN and 212LN cells were transfected with vector, p70S6K wild type constructs, or treated with 50 ng/ml IL-6, as indicated, for 24 hours. Then cells were subjected to a transwell assay. Magnification: 100×. Columns, means of cell number in five selected fields; bars, SD. *p < 0.05.

Figure 4. Downregulation of p70S6K expression inhibits IL-6-induced EMT and migration

(A) 686LN cells were transfected with p70S6K siRNAs or control siRNAs for 24 hours, then they were serum starved overnight and treated with 50 ng/ml IL-6 for another 48 hours. (B and C) stable 686LN cell lines with p70S6K knockdown (p70S6K_shRNA) or the control cell lines (scramble_shRNA) were harvested directly (B) or harvested after being serum starved overnight and subsequently treated with IL-6 for 48 hours (C). Whole-cell protein lysates were prepared and subjected to western blotting. The fold change of each treatment vs. the control was calculated after quantification and presented under each blot. (D) transwell assay of stable cell lines, previously mentioned, with or without 50 ng/ml IL-6 for 24 hours. Magnification: 100×. Columns, means of cell number in five selected fields; bars, SD. *p < 0.05, ^#p > 0.05.

Figure 5. IL-6-induced activation of p70S6K locates downstream of the PI3K/Akt/mTOR and MAPK signaling pathways

(A) 686LN cells were serum starved overnight and treated with 50 ng/ml IL-6 for different times, as indicated. (B, C, and D) 686LN cells were serum starved overnight and pretreated with different inhibitors: wortmannin 250 nmol/L (B), LY294002 20 μmol/L (B), rapamycin 100 nmol/L (C), or U0126 5 μmol/L (D) for 30 minutes, then co-treated with or without IL-6 50 ng/ml for another hour. Whole-cell protein lysates were prepared and subjected to western blotting. C, control; W, wortmannin; LY, LY294002.

Figure 6. IL-6-induced activation of p70S6K locates downstream of STAT3

(A) 686LN cells were transfected with two different sequences of STAT3 siRNAs (STAT-1 and STAT-2) or control siRNAs for 24 hours, then they were serum starved overnight and treated with or without 50 ng/ml IL-6 for another hour. (B) 686LN cells were serum starved overnight and pretreated with niclosamide 1 μmol/L for 30 minutes, then co-treated with or without IL-6 50 ng/ml for another hour. (C) 686LN and 212LN cells were transfected with p70S6K pool siRNAs (four sequences) or control siRNAs for 24 hours, then were serum starved overnight and treated with or without IL-6 50 ng/ml for another hour. Whole-cell protein lysates were prepared and subjected to western blot analysis. L.E., long exposure.

Figure 7. A diagram of IL-6-induced activation of p70S6K in the metastasis of HNSCCs

IL-6 activated the PI3K/Akt/mTOR, MAPK, and STAT3 signaling pathways. p70S6K located downstream of the PI3K/Akt/mTOR, MAPK, and STAT3 signaling pathways, because wortmannin, LY294002, rapamycin, U0126, and niclosamide inhibited IL-6-induced activation of p70S6K. p70S6K was a node for multiple signaling pathways to mediate IL-6-induced EMT and the metastasis of HNSCCs.